Anti-OX40 antibodies and diagnostic uses thereof

ABSTRACT

The invention provides antibodies immunoreactive with human OX40 and methods of using the same. The antibodies are reactive with a portion of the C-terminus of the human OX40 protein that includes amino acids 266-277. The antibodies are useful for detecting OX40 protein expression in human tissue samples, including by immunohistochemistry, immunofluorescence, or immunoblot.

CROSS-REFERENCE TO RELATED APPLICATIONS

This is a continuation of PCT/EP2016/072236, filed Sep. 20, 2016, andclaims the benefit of U.S. Provisional Patent Application 62/222,105,filed Sep. 22, 2015, the content of each of which is incorporated byreference in its entirety.

SEQUENCE LISTING INCORPORATION BY REFERENCE

A sequence listing submitted herewith in a computer-readable format,having a file name of 33058US1_ST25.txt, created on Mar. 13, 2018, whichis 8,570 bytes in size, is hereby incorporated-by-reference.

BACKGROUND OF THE INVENTION Field of the Invention

The present invention relates to antibodies reactive with human OX40(anti-OX40) and methods of using the same.

Brief Description of Related Art

OX40 is a 277 amino acid single pass type I membrane protein thatfunctions as a receptor for Tumor necrosis factor ligand superfamilymember 4 ligand (also called OX40L and CD252). Activation of OX40 hasbeen shown increase their proliferation, survival, and effector functionof cytotoxic T-lymphocytes. Curti et al., Cancer Res., Vol. 73, Issue24, pp. 7189-98 (Oct. 31, 2013). Clinical trials are currently underwaytesting anti-OX40 antibodies in patients with advanced cancers todetermine whether it is a useful target for potentiation of anti-tumorimmune responses.

SUMMARY

The present disclosure relates to anti-OX40 antibodies and methods ofusing the same.

In one aspect, an antibody, antigen-binding fragment thereof, or arecombinant protein thereof is disclosed, wherein the antibody iscapable of specifically binding to OX40.

In one aspect, an antibody, antigen-binding fragment thereof, or arecombinant protein thereof is disclosed, wherein the antibody iscapable of specifically binding to amino acids 266-277 of SEQ ID NO: 1.

In one aspect, an antibody, antigen-binding fragment thereof, or arecombinant protein thereof is disclosed, wherein the antibody iscapable of specifically binding to OX40, wherein the antibody binds toan epitope comprising 266-277 of human OX40 polypeptide (SEQ ID NO: 1).In some embodiments, the antibody comprises the following hypervariableregions (HVRs): (a) an HVR-H1 comprising the amino acid sequence ofSDNIQ (SEQ ID NO: 2); (b) an HVR-H2 comprising the amino acid sequenceof AVDYNNKPFYANWAKG (SEQ ID NO: 3); and (c) an HVR-H3 comprising theamino acid sequence of NTFSP (SEQ ID NO: 4). In some embodiments, theantibody further comprises the following heavy chain variable domainframework regions (FRs): (a) FR-H1 comprising the amino acid sequence ofQSLEESGGRLVAPGGSLTLTCTVSGIDLS (SEQ ID NO: 5); (b) FR-H2 comprising theamino acid sequence of WVRQAPGKGLEWIG (SEQ ID NO: 6); (c) FR-H3comprising the amino acid sequence of RFTISKTSSTTVDLKMTSLTTEDTATYFCAK(SEQ ID NO: 7); and (d) FR-H4 comprising the amino acid sequence ofWGPGTLVTVSS (SEQ ID NO: 8). In some embodiments, the antibody furthercomprises the following HVRs: (a) an HVR-L1 comprising the amino acidsequence of QSSQSVYNANHLS (SEQ ID NO: 9); (b) an HVR-L2 comprising theamino acid sequence of YISTPDS (SEQ ID NO: 10); and (c) an HVR-L3comprising the amino acid sequence of CAALNSDEVFT (SEQ ID NO: 11). Insome embodiments, the antibody further comprises the following lightchain variable domain FRs: (a) FR-L1 comprising the amino acid sequenceof DPAMTQTPSSTSAAVGGTVTINC (SEQ ID NO: 12); (b) FR-L2 comprising theamino acid sequence of WFQQKPGQPPKRLIY (SEQ ID NO: 13); (c) FR-L3comprising the amino acid sequence of GVPPRFSGSGSGTQFTLTISGVQCDDAATYY(SEQ ID NO: 14); and (d) FR-L4 comprising the amino acid sequence ofFGGGTEVVVK (SEQ ID NO: 15). In some embodiments, the antibody comprises(a) a VH sequence having at least 95% sequence identity to the aminoacid sequence of SEQ ID NO: 16; (b) a VL sequence having at least 95%sequence identity to the amino acid sequence of SEQ ID NO: 17; or (c) aVH sequence as in (a) and a VL sequence as in (b). In some embodiments,the antibody comprises a VH sequence of SEQ ID NO: 16. In someembodiments, the antibody comprises a VL sequence of SEQ ID NO: 17.

In other embodiments, the antibody comprises the following HVRs: (a) anHVR-L1 comprising the amino acid sequence of SEQ ID NO: 9; (b) an HVR-L2comprising the amino acid sequence of SEQ ID NO: 10; and (c) an HVR-L3comprising the amino acid sequence of SEQ ID NO: 11. In someembodiments, the antibody further comprises the following light chainvariable domain FRs: (a) FR-L1 comprising the amino acid sequence of SEQID NO: 12; (b) FR-L2 comprising the amino acid sequence of SEQ ID NO:13; (c) FR-L3 comprising the amino acid sequence of SEQ ID NO: 14; and(d) FR-L4 comprising the amino acid sequence of SEQ ID NO: 15.

In another aspect, the invention features an isolated antibody thatspecifically binds OX40, wherein the antibody comprises the followingHVRs: (a) an HVR-H1 comprising the amino acid sequence of SEQ ID NO: 2;(b) an HVR-H2 comprising the amino acid sequence of SEQ ID NO: 3; (c) anHVR-H3 comprising the amino acid sequence of SEQ ID NO: 4; (d) an HVR-L1comprising the amino acid sequence of SEQ ID NO: 9; (e) an HVR-L2comprising the amino acid sequence of SEQ ID NO: 10; and (f) an HVR-L3comprising the amino acid sequence of SEQ ID NO: 11. In someembodiments, the antibody further comprises the following heavy chainvariable domain and light chain variable domain FRs: (a) FR-H1comprising the amino acid sequence of SEQ ID NO: 5; (b) FR-H2 comprisingthe amino acid sequence of SEQ ID NO: 6; (c) FR-H3 comprising the aminoacid sequence of SEQ ID NO: 7; (d) FR-H4 comprising the amino acidsequence of SEQ ID NO: 8; (e) FR-L1 comprising the amino acid sequenceof SEQ ID NO: 12; (f) FR-L2 comprising the amino acid sequence of SEQ IDNO: 13; (g) FR-L3 comprising the amino acid sequence of SEQ ID NO: 14;and (h) FR-L4 comprising the amino acid sequence of SEQ ID NO: 15. Insome embodiments, the antibody comprises a VH sequence of SEQ ID NO: 16and a VL sequence of SEQ ID NO: 17.

In another aspect, the invention features an isolated antibody thatcompetes for binding to OX40 with any one of the preceding antibodies.

In another aspect, the invention features an isolated antibody thatbinds to the same epitope as any one of the preceding antibodies.

In some embodiments, any one of the preceding antibodies can be amonoclonal antibody. In some embodiments, the monoclonal antibody can bea rabbit monoclonal antibody.

In some embodiments, any one of the preceding antibodies can be an IgGantibody (e.g., an IgG1 antibody).

In some embodiments, any one of the preceding antibodies can be anantibody fragment that specifically binds OX40. In some embodiments, theantibody fragment is selected from the group consisting of Fab, singlechain variable fragment (scFv), Fv, Fab′, Fab′-SH, F(ab′)2, and diabody.

In another aspect, the invention features an immunoconjugate comprisingany one of the preceding antibodies.

In another aspect, the invention features an isolated nucleic acid thatencodes any of the antibodies described herein. In another aspect, theinvention features a vector (e.g., an expression vector) comprising thenucleic acid for expressing the antibody. In another aspect, theinvention features host cells comprising the preceding nucleic acidsand/or vectors.

In some aspects, any one of the preceding antibodies can be for use indetecting the presence or expression level of OX40 in a biologicalsample. In some embodiments, the detecting is by immunohistochemistry(IHC), immunofluorescence (IF), or immunoblot. In some embodiments, thedetecting is by IHC. In some embodiments, the sample comprises a fixedtissue. In some embodiments, the fixed tissue is a formalin-fixedparaffin-embedded (FFPE) tissue. In some embodiments, the sample is froma subject having, or predisposed to, cancer or an autoimmune disease.

A further aspect of the invention is a method of detecting the presenceor expression level of OX40 in a biological sample comprising contactingthe biological sample with any one of the preceding antibodies anddetecting the presence of the bound antibody. In some embodiments, thedetecting is by IHC, IF, or immunoblot. In some embodiments, thedetecting is by IHC. In some embodiments, the sample comprises a fixedtissue. In some embodiments, the fixed tissue is a FFPE tissue. In someembodiments, the sample is from a subject having or predisposed tocancer or autoimmune disease.

BRIEF DESCRIPTION OF THE DRAWINGS

The application file contains at least one drawing executed in color.Copies of this patent or patent application with color drawings will beprovided by the Office upon request and payment of the necessary fee.

FIG. 1 is a schematic diagram showing the general antibody productionprocess for the anti-OX40 antibody.

FIG. 2 is an image showing the results of immunohistochemistry (IHC) onformalin-fixed, paraffin-Embedded (FFPE) cells as follows: (A) Mocktransfected cells (negative control cells); (B) OX-40 transfected cells(positive control cells); (C) Reactive lymph node; and (D) Prostateadenocarcinoma.

DETAILED DESCRIPTION OF EMBODIMENTS OF THE INVENTION I. Definitions

The terms “anti-OX40 antibody,” “anti-OX40 antibody,” “antibody thatspecifically binds to OX40,” and “antibody that binds to OX40” refer toan antibody that is capable of binding OX40 with sufficient affinitysuch that the antibody is useful as a diagnostic and/or therapeuticagent in targeting OX40. In one embodiment, the extent of binding of ananti-OX40 antibody to an unrelated, non-OX40 protein is less than about10% of the binding of the antibody to OX40 as measured, e.g., by aradioimmunoassay (RIA). In certain embodiments, an antibody that bindsto OX40 has a dissociation constant (Kd) of ≤1 μM, ≤100 nM, ≤10 nM, ≤1nM, ≤0.1 nM, ≤0.01 nM, or ≤0.001 nM (e.g., 10⁻⁸M or less, e.g., from10⁻⁸ M to 10⁻¹³ M, e.g., from 10⁻⁹M to 10⁻¹³ M). In certain embodiments,an anti-OX40 antibody binds to an epitope of OX40 that is conservedamong OX40 from different species.

The term “antibody” herein is used in the broadest sense and encompassesvarious antibody structures, including but not limited to monoclonalantibodies, polyclonal antibodies, multispecific antibodies (e.g.,bispecific antibodies), and antibody fragments so long as they exhibitthe desired antigen-binding activity.

An “antibody fragment” refers to a molecule other than an intactantibody that comprises a portion of an intact antibody that binds theantigen to which the intact antibody binds. Examples of antibodyfragments include but are not limited to Fv, Fab, Fab′, Fab′-SH,F(ab′)₂; diabodies; linear antibodies; single-chain antibody molecules(e.g. scFv); and multispecific antibodies formed from antibodyfragments.

An “antibody that binds to the same epitope” as a reference antibodyrefers to an antibody that blocks binding of the reference antibody toits antigen in a competition assay by 50% or more, and conversely, thereference antibody blocks binding of the antibody to its antigen in acompetition assay by 50% or more. An exemplary competition assay isprovided herein.

An “autoimmune disease” is a disease or disorder arising from anddirected against an individual's own tissues or organs or aco-segregation or manifestation thereof or resulting conditiontherefrom. Autoimmune diseases can be an organ-specific disease (i.e.,the immune response is specifically directed against an organ systemsuch as the endocrine system, the hematopoietic system, the skin, thecardiopulmonary system, the gastrointestinal and liver systems, therenal system, the thyroid, the ears, the neuromuscular system, thecentral nervous system, etc.) or a systemic disease that can affectmultiple organ systems (for example, systemic lupus erythematosus (SLE),rheumatoid arthritis (RA), polymyositis, etc.). Non-limiting exemplaryautoimmune diseases include autoimmune rheumatologic disorders (such as,for example, RA, Sjogren's syndrome, scleroderma, lupus such as SLE andlupus nephritis, polymyositis-dermatomyositis, cryoglobulinemia,anti-phospholipid antibody syndrome, and psoriatic arthritis),autoimmune gastrointestinal and liver disorders (such as, for example,inflammatory bowel diseases (e.g., ulcerative colitis and Crohn'sdisease), autoimmune gastritis and pernicious anemia, autoimmunehepatitis, primary biliary cirrhosis, primary sclerosing cholangitis,and celiac disease), vasculitis (such as, for example, ANCA-negativevasculitis and ANCA-associated vasculitis, including Churg-Straussvasculitis, Wegener's granulomatosis, and microscopic polyangiitis),autoimmune neurological disorders (such as, for example, multiplesclerosis, opsoclonus myoclonus syndrome, myasthenia gravis,neuromyelitis optica, Parkinson's disease, Alzheimer's disease, andautoimmune polyneuropathies), renal disorders (such as, for example,glomerulonephritis, Goodpasture's syndrome, and Berger's disease),autoimmune dermatologic disorders (such as, for example, psoriasis,urticaria, hives, pemphigus vulgaris, bullous pemphigoid, and cutaneouslupus erythematosus), hematologic disorders (such as, for example,thrombocytopenic purpura, thrombotic thrombocytopenic purpura,post-transfusion purpura, and autoimmune hemolytic anemia),atherosclerosis, uveitis, autoimmune hearing diseases (such as, forexample, inner ear disease and hearing loss), Behcet's disease,Raynaud's syndrome, organ transplant, and autoimmune endocrine disorders(such as, for example, diabetic-related autoimmune diseases such asinsulin-dependent diabetes mellitus (IDDM), Addison's disease, andautoimmune thyroid disease (e.g., Graves' disease and thyroiditis)).More preferred such diseases include, for example, RA, ulcerativecolitis, ANCA-associated vasculitis, lupus, multiple sclerosis,Sjogren's syndrome, Graves' disease, IDDM, pernicious anemia,thyroiditis, and glomerulonephritis.

By “biological sample” is meant a collection of similar cells obtainedfrom a subject or patient. A biological sample can be a tissue or a cellsample. The source of the tissue or cell sample may be solid tissue asfrom a fresh, frozen and/or preserved organ or tissue sample or biopsyor aspirate; blood or any blood constituents; bodily fluids such ascerebral spinal fluid, amniotic fluid, peritoneal fluid, or interstitialfluid; cells from any time in gestation or development of the subject.The biological sample can also be obtained from in vitro tissue or cellculture. The tissue sample may contain compounds which are not naturallyintermixed with the tissue in nature such as preservatives,anticoagulants, buffers, fixatives, nutrients, antibiotics, or the like.Examples of biological samples herein include, but are not limited to,tumor biopsies, circulating tumor cells, serum or plasma, circulatingplasma proteins, ascitic fluid, primary cell cultures or cell linesderived from tumors or exhibiting tumor-like properties, as well aspreserved tumor samples, such as formalin-fixed, paraffin-embedded tumorsamples or frozen tumor samples.

The terms “cancer” and “cancerous” refer to or describe thephysiological condition in mammals that is typically characterized byunregulated cell growth/proliferation. Examples of cancer include, butare not limited to, carcinoma, lymphoma (e.g., Hodgkin's andnon-Hodgkin's lymphoma), blastoma, sarcoma, and leukemia. Moreparticular examples of such cancers include squamous cell cancer,small-cell lung cancer, non-small cell lung cancer, adenocarcinoma ofthe lung, squamous carcinoma of the lung, cancer of the peritoneum,hepatocellular cancer, gastrointestinal cancer, pancreatic cancer,glioma, cervical cancer, ovarian cancer, liver cancer, bladder cancer,hepatoma, breast cancer, colon cancer, colorectal cancer, endometrial oruterine carcinoma, salivary gland carcinoma, kidney cancer, livercancer, prostate cancer, vulval cancer, thyroid cancer, hepaticcarcinoma, leukemia and other lymphoproliferative disorders, and varioustypes of head and neck cancer.

The term “chimeric” antibody refers to an antibody in which a portion ofthe heavy and/or light chain is derived from a particular source orspecies, while the remainder of the heavy and/or light chain is derivedfrom a different source or species.

The “class” of an antibody refers to the type of constant domain orconstant region possessed by its heavy chain. There are five majorclasses of antibodies: IgA, IgD, IgE, IgG, and IgM, and several of thesemay be further divided into subclasses (isotypes), e.g., IgG₁, IgG₂,IgG₃, IgG₄, IgA₁, and IgA₂. The heavy chain constant domains thatcorrespond to the different classes of immunoglobulins are called α, δ,ε, γ, and μ respectively.

The term “cytotoxic agent” as used herein refers to a substance thatinhibits or prevents a cellular function and/or causes cell death ordestruction. Cytotoxic agents include, but are not limited to,radioactive isotopes (e.g., At²¹¹, I¹³¹, I¹²⁵, Y⁹⁰, Re¹⁸⁶, Re¹⁸⁸, Sm¹⁵³,Bi²¹², P³², Pb²¹² and radioactive isotopes of Lu); chemotherapeuticagents or drugs (e.g., methotrexate, adriamicin, vinca alkaloids(vincristine, vinblastine, etoposide), doxorubicin, melphalan, mitomycinC, chlorambucil, daunorubicin or other intercalating agents); growthinhibitory agents; enzymes and fragments thereof such as nucleolyticenzymes; antibiotics; toxins such as small molecule toxins orenzymatically active toxins of bacterial, fungal, plant or animalorigin, including fragments and/or variants thereof; and the variousantitumor or anticancer agents disclosed below.

“Effector functions” refer to those biological activities attributableto the Fc region of an antibody, which vary with the antibody isotype.Examples of antibody effector functions include: C1q binding andcomplement dependent cytotoxicity (CDC); Fc receptor binding;antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (e.g. B cell receptor); and B cellactivation.

The term “Fc region” herein is used to define a C-terminal region of animmunoglobulin heavy chain that contains at least a portion of theconstant region. The term includes native sequence Fc regions andvariant Fc regions. In one embodiment, a human IgG heavy chain Fc regionextends from Cys226, or from Pro230, to the carboxyl-terminus of theheavy chain. However, the C-terminal lysine (Lys447) of the Fc regionmay or may not be present. Unless otherwise specified herein, numberingof amino acid residues in the Fc region or constant region is accordingto the EU numbering system, also called the EU index, as described inKabat et al. Sequences of Proteins of Immunological Interest. 5th Ed.Public Health Service, National Institutes of Health, Bethesda, Md.,1991.

“Framework” or “FR” refers to variable domain residues other thanhypervariable region (HVR) residues. The FR of a variable domaingenerally consists of four FR domains: FR1, FR2, FR3, and FR4.Accordingly, the HVR and FR sequences generally appear in the followingsequence in VH (or VL): FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4.

The terms “full-length antibody,” “intact antibody,” and “wholeantibody” are used herein interchangeably to refer to an antibody havinga structure substantially similar to a native antibody structure orhaving heavy chains that contain an Fc region as defined herein.

The terms “level of expression” or “expression level” in general areused interchangeably and generally refer to the amount of apolynucleotide, mRNA, or an amino acid product or protein in abiological sample. “Expression” generally refers to the process by whichgene-encoded information is converted into the structures present andoperating in the cell. Therefore, according to the invention“expression” of a gene (e.g., the OX40 gene) may refer to transcriptioninto a polynucleotide, translation into a protein, or evenposttranslational modification of the protein. Fragments of thetranscribed polynucleotide, the translated protein, or thepost-translationally modified protein shall also be regarded asexpressed whether they originate from a transcript generated byalternative splicing or a degraded transcript, or from apost-translational processing of the protein, e.g., by proteolysis. Insome embodiments, “expression level” refers to amount of a protein(e.g., OX40) in a biological sample as determined usingimmunohistochemistry (IHC), immunoblotting (e.g., Western blotting),immunofluorescence (IF), Enzyme-Linked Immunosorbant Assay (ELISA), orflow cytometry.

The terms “host cell,” “host cell line,” and “host cell culture” areused interchangeably and refer to cells into which exogenous nucleicacid has been introduced, including the progeny of such cells. Hostcells include “transformants” and “transformed cells”, which include theprimary transformed cell and progeny derived therefrom without regard tothe number of passages. Progeny may not be completely identical innucleic acid content to a parent cell, but may contain mutations. Mutantprogeny that have the same function or biological activity as screenedor selected for in the originally transformed cell are included herein.

A “human antibody” is one which possesses an amino acid sequence whichcorresponds to that of an antibody produced by a human or a human cellor derived from a non-human source that utilizes human antibodyrepertoires or other human antibody-encoding sequences. This definitionof a human antibody specifically excludes a humanized antibodycomprising non-human antigen-binding residues.

A “human consensus framework” is a framework which represents the mostcommonly occurring amino acid residues in a selection of humanimmunoglobulin VL or VH framework sequences. Generally, the selection ofhuman immunoglobulin VL or VH sequences is from a subgroup of variabledomain sequences. Generally, the subgroup of sequences is a subgroup asin Kabat et al., Sequences of Proteins of Immunological Interest. FifthEdition, NIH Publication 91-3242, Bethesda Md., Vols. 1-3, 1991. In oneembodiment, for the VL, the subgroup is subgroup kappa I as in Kabat etal., supra. In one embodiment, for the VH, the subgroup is subgroup IIIas in Kabat et al., supra.

A “humanized” antibody refers to a chimeric antibody comprising aminoacid residues from non-human HVRs and amino acid residues from humanFRs. In certain embodiments, a humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the HVRs (e.g., CDRs) correspond tothose of a non-human antibody, and all or substantially all of the FRscorrespond to those of a human antibody. A humanized antibody optionallymay comprise at least a portion of an antibody constant region derivedfrom a human antibody. A “humanized form” of an antibody, e.g., anon-human antibody, refers to an antibody that has undergonehumanization.

The term “hypervariable region” or “HVR” as used herein refers to eachof the regions of an antibody variable domain which are hypervariable insequence (“complementarity determining regions” or “CDRs”) and/or formstructurally defined loops (“hypervariable loops”) and/or contain theantigen-contacting residues (“antigen contacts”). Generally, antibodiescomprise six HVRs: three in the VH (H1, H2, H3), and three in the VL(L1, L2, L3). Exemplary HVRs herein include:

-   -   (a) hypervariable loops occurring at amino acid residues 26-32        (L1), 50-52 (L2), 91-96 (L3), 26-32 (H1), 53-55 (H2), and 96-101        (H3) (Chothia et al. J. Mol. Biol. 196: 901-917, 1987);    -   (b) CDRs occurring at amino acid residues 24-34 (L1), 50-56        (L2), 89-97 (L3), 31-35b (H1), 50-65 (H2), and 95-102 (H3)        (Kabat et al., Sequences of Proteins of Immunological Interest.        5th Ed. Public Health Service, National Institutes of Health,        Bethesda, Md., 1991);    -   (c) antigen contacts occurring at amino acid residues 27c-36        (L1), 46-55 (L2), 89-96 (L3), 30-35b (H1), 47-58 (H2), and        93-101 (H3) (MacCallum et al. J. Mol. Biol. 262: 732-745, 1996);        and    -   (d) combinations of (a), (b), and/or (c), including HVR amino        acid residues 46-56 (L2), 47-56 (L2), 48-56 (L2), 49-56 (L2),        26-35 (H1), 26-35b (H1), 49-65 (H2), 93-102 (H3), and 94-102        (H3). Unless otherwise indicated, HVR residues and other        residues in the variable domain (e.g., FR residues) are numbered        herein according to Kabat et al., supra.

An “immunoconjugate” is an antibody conjugated to one or moreheterologous molecule(s), including but not limited to a cytotoxicagent.

An “isolated” antibody is one which has been separated from a componentof its natural environment. In some embodiments, an antibody is purifiedto greater than 95% or 99% purity as determined by, for example,electrophoretic (e.g., SDS-PAGE, isoelectric focusing (IEF), capillaryelectrophoresis) or chromatographic (e.g., ion exchange or reverse phaseHPLC). For review of methods for assessment of antibody purity, see,e.g., Flatman et al. J. Chromatogr. B. 848: 79-87, 2007.

An “isolated” nucleic acid refers to a nucleic acid molecule that hasbeen separated from a component of its natural environment. An isolatednucleic acid includes a nucleic acid molecule contained in cells thatordinarily contain the nucleic acid molecule, but the nucleic acidmolecule is present extrachromosomally or at a chromosomal location thatis different from its natural chromosomal location.

“Isolated nucleic acid encoding an anti-OX40 antibody” refers to one ormore nucleic acid molecules encoding antibody heavy and light chains (orfragments thereof), including such nucleic acid molecule(s) in a singlevector or separate vectors, and such nucleic acid molecule(s) present atone or more locations in a host cell.

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicaland/or bind the same epitope, except for possible variant antibodies,e.g., containing naturally occurring mutations or arising duringproduction of a monoclonal antibody preparation, such variants generallybeing present in minor amounts. In contrast to polyclonal antibodypreparations, which typically include different antibodies directedagainst different determinants (epitopes), each monoclonal antibody of amonoclonal antibody preparation is directed against a single determinanton an antigen. Thus, the modifier “monoclonal” indicates the characterof the antibody as being obtained from a substantially homogeneouspopulation of antibodies, and is not to be construed as requiringproduction of the antibody by any particular method. For example, themonoclonal antibodies to be used in accordance with the presentinvention may be made by a variety of techniques, including but notlimited to the hybridoma method, recombinant DNA methods, phage-displaymethods, and methods utilizing transgenic animals containing all or partof the human immunoglobulin loci, or a combination thereof.

“Percent (%) amino acid sequence identity” with respect to a referencepolypeptide sequence is defined as the percentage of amino acid residuesin a candidate sequence that are identical with the amino acid residuesin the reference polypeptide sequence, after aligning the sequences andintroducing gaps, if necessary, to achieve the maximum percent sequenceidentity, and not considering any conservative substitutions as part ofthe sequence identity. Alignment for purposes of determining percentamino acid sequence identity can be achieved in various ways that arewithin the skill in the art, for instance, using publicly availablecomputer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR)software. Those skilled in the art can determine appropriate parametersfor aligning sequences, including any algorithms needed to achievemaximal alignment over the full length of the sequences being compared.For purposes herein, however, % amino acid sequence identity values aregenerated using the sequence comparison computer program ALIGN-2. TheALIGN-2 sequence comparison computer program was authored by Genentech,Inc., and the source code has been filed with user documentation in theU.S. Copyright Office, Washington D.C., 20559, where it is registeredunder U.S. Copyright Registration No. TXU510087. The ALIGN-2 program ispublicly available from Genentech, Inc., South San Francisco, Calif., ormay be compiled from the source code. The ALIGN-2 program should becompiled for use on a UNIX operating system, including digital UNIXV4.0D. All sequence comparison parameters are set by the ALIGN-2 programand do not vary.

In situations where ALIGN-2 is employed for amino acid sequencecomparisons, the % amino acid sequence identity of a given amino acidsequence A to, with, or against a given amino acid sequence B (which canalternatively be phrased as a given amino acid sequence A that has orcomprises a certain % amino acid sequence identity to, with, or againsta given amino acid sequence B) is calculated as follows:100 times the fraction X/Ywhere X is the number of amino acid residues scored as identical matchesby the sequence alignment program ALIGN-2 in that program's alignment ofA and B, and where Y is the total number of amino acid residues in B. Itwill be appreciated that where the length of amino acid sequence A isnot equal to the length of amino acid sequence B, the % amino acidsequence identity of A to B will not equal the % amino acid sequenceidentity of B to A. Unless specifically stated otherwise, all % aminoacid sequence identity values used herein are obtained as described inthe immediately preceding paragraph using the ALIGN-2 computer program.

The term “OX40,” as used herein, refers to any native OX40 from anyvertebrate source, including mammals such as primates (e.g., humans) androdents (e.g., mice and rats), unless otherwise indicated. The termencompasses “full-length,” unprocessed OX40 as well as any form of OX40that results from processing in the cell. The term also encompassesnaturally occurring variants of OX40, e.g., splice variants or allelicvariants. The amino acid sequence of an exemplary full-length human OX40protein is shown in SEQ ID NO: 1. The amino acid sequence of anexemplary full-length human OX40 protein can be found, e.g., underUniProt Accession No. P43489.

As use herein, the term “specifically binds to” or is “specific for”refers to measurable and reproducible interactions such as bindingbetween a target and an antibody, which is determinative of the presenceof the target in the presence of a heterogeneous population of moleculesincluding biological molecules. For example, an antibody thatspecifically binds to a target (which can be an epitope, e.g., aminoacid residues 266-277 of SEQ ID NO: 1) is an antibody that binds thistarget with greater affinity, avidity, more readily, and/or with greaterduration than it binds to other targets. In one embodiment, the extentof binding of an antibody to an unrelated target is less than about 10%of the binding of the antibody to the target as measured, e.g., by aradioimmunoassay (RIA). In certain embodiments, an antibody thatspecifically binds to a target has a dissociation constant (Kd) of ≤1μM, ≤100 nM, ≤10 nM, ≤1 nM, or ≤0.1 nM. In certain embodiments, anantibody specifically binds to an epitope on a protein that is conservedamong the protein from different species. In another embodiment,specific binding can include, but does not require exclusive binding.

A “subject” or “individual” is a mammal. Mammals include, but are notlimited to, domesticated animals (e.g., cows, sheep, cats, dogs, andhorses), primates (e.g., humans and non-human primates such as monkeys),rabbits, and rodents (e.g., mice and rats). In certain embodiments, theindividual or subject is a human.

The term “variable region” or “variable domain” refers to the domain ofan antibody heavy or light chain that is involved in binding theantibody to antigen. The variable domains of the heavy chain and lightchain (VH and VL, respectively) of a native antibody generally havesimilar structures, with each domain comprising four conserved frameworkregions (FRs) and three hypervariable regions (HVRs). See, e.g., Kindtet al. Kuby Immunology. 6^(th) ed., page 91, W.H. Freeman and Co., 2007.A single VH or VL domain may be sufficient to confer antigen-bindingspecificity. Furthermore, antibodies that bind a particular antigen maybe isolated using a VH or VL domain from an antibody that binds theantigen to screen a library of complementary VL or VH domains,respectively. See, e.g., Portolano et al. J. Immunol. 150: 880-887, 1993and Clarkson et al. Nature. 352: 624-628, 1991.

The term “vector,” as used herein, refers to a nucleic acid moleculecapable of propagating another nucleic acid to which it is linked. Theterm includes the vector as a self-replicating nucleic acid structure aswell as the vector incorporated into the genome of a host cell intowhich it has been introduced. Certain vectors are capable of directingthe expression of nucleic acids to which they are operatively linked.Such vectors are referred to herein as “expression vectors”.

II. Compositions and Methods

The invention provides novel antibodies that bind to OX40. Antibodies ofthe invention are useful, for example, for detecting the presence ofOX40 or the expression level of OX40 (e.g., in biological samples).

A. Exemplary Anti-OX40 Antibodies

The invention provides anti-OX40 antibodies useful for, e.g., diagnosticapplications (e.g., immunohistochemistry (IHC), immunofluorescence (IF),and immunoblot (e.g., Western blot)). In one example, the inventionprovides anti-OX40 antibodies that bind to an a C-terminal epitope ofOX40 (e.g., amino acid residues 266-277 SEQ ID NO: 1). The epitope onOX40 may be recognized in a manner that is conformation-dependent orconformation-independent.

In some instances, the anti-OX40 antibodies that bind to amino acidresidues 266-277 of OX40 include at least one, two, three, four, five,or six HVRs selected from (a) HVR-H1 comprising the amino acid sequenceof SEQ ID NO: 2; (b) HVR-H2 comprising the amino acid sequence of SEQ IDNO: 3; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 4;(d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 9; (e)HVR-L2 comprising the amino acid sequence of SEQ ID NO: 10; and (f)HVR-L3 comprising the amino acid sequence of SEQ ID NO: 11. For example,in some instances, the anti-OX40 antibodies include (a) an HVR-H1comprising the amino acid sequence of SEQ ID NO: 2; (b) an HVR-H2comprising the amino acid sequence of SEQ ID NO: 3; and (c) an HVR-H3comprising the amino acid sequence of SEQ ID NO: 4. In some instances,the anti-OX40 antibodies include (a) an HVR-L1 comprising the amino acidsequence of SEQ ID NO: 9; (b) HVR-L2 comprising the amino acid sequenceof SEQ ID NO: 10; and (c) HVR-L3 comprising the amino acid sequence ofSEQ ID NO: 11.

In some instances wherein the anti-OX40 antibodies bind to amino acidresidues 266-277 of OX40 and include (a) an HVR-H1 comprising the aminoacid sequence of SEQ ID NO: 2; (b) an HVR-H2 comprising the amino acidsequence of SEQ ID NO: 3; and (c) an HVR-H3 comprising the amino acidsequence of SEQ ID NO: 4, the anti-OX40 antibodies further include thefollowing heavy chain variable domain framework regions (FRs): (a) FR-H1comprising the amino acid sequence of SEQ ID NO: 5; (b) FR-H2 comprisingthe amino acid sequence of SEQ ID NO: 6; (c) FR-H3 comprising the aminoacid sequence of SEQ ID NO: 7; or (d) FR-H4 comprising the amino acidsequence of SEQ ID NO: 8. In some instances wherein the anti-OX40antibodies bind to amino acid residues 266-277 of OX40 and include (a)an HVR-H1 comprising the amino acid sequence of SEQ ID NO: 2; (b) anHVR-H2 comprising the amino acid sequence of SEQ ID NO: 3; and (c) anHVR-H3 comprising the amino acid sequence of SEQ ID NO: 4, the anti-OX40antibodies further include the following heavy chain variable domainframework regions (FRs): (a) FR-H1 comprising the amino acid sequence ofSEQ ID NO: 5; (b) FR-H2 comprising the amino acid sequence of SEQ ID NO:6; (c) FR-H3 comprising the amino acid sequence of SEQ ID NO: 7; and (d)FR-H4 comprising the amino acid sequence of SEQ ID NO: 8.

In some instances wherein the anti-OX40 antibodies bind to amino acidresidues 266-277 of OX40, the antibodies include (a) an HVR-H1comprising the amino acid sequence of SEQ ID NO: 2; (b) an HVR-H2comprising the amino acid sequence of SEQ ID NO: 3; (c) an HVR-H3comprising the amino acid sequence of SEQ ID NO: 4; (d) an HVR-L1comprising the amino acid sequence of SEQ ID NO: 9; (e) an HVR-L2comprising the amino acid sequence of SEQ ID NO: 10; and (f) an HVR-L3comprising the amino acid sequence of SEQ ID NO: 11. In some instances,these anti-OX40 antibodies include the following FRs: (a) FR-H1comprising the amino acid sequence of SEQ ID NO: 5; (b) FR-H2 comprisingthe amino acid sequence of SEQ ID NO: 6; (c) FR-H3 comprising the aminoacid sequence of SEQ ID NO: 7; and (d) FR-H4 comprising the amino acidsequence of SEQ ID NO: 8 and may additionally or alternatively include(e) FR-L1 comprising the amino acid sequence SEQ ID NO: 12; (f) FR-L2comprising the amino acid sequence of SEQ ID NO: 13; (g) FR-L3comprising the amino acid sequence of SEQ ID NO: 14; and (h) FR-L4comprising the amino acid sequence of SEQ ID NO: 15.

In some instances, the anti-OX40 antibodies that bind to amino acidresidues 266-277 of OX40 may also include a heavy chain variable domain(VH) sequence having at least 80% (e.g., at least 81%, 82%, 83%, 84%,85%, 86%, 87%, 88%, or 89%), at least 90% (e.g., at least 91%, 92%, 93%,or 94%), or at least 95% (e.g., at least 96%, 97%, 98%, or 99%) sequenceidentity to, or the sequence of, the amino acid sequence of SEQ ID NO:16. In certain embodiments, a VH sequence having at least 80%, 81%, 82%,83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, or 99% identity contains substitutions (e.g., conservativesubstitutions), insertions, or deletions relative to the referencesequence (SEQ ID NO: 16), but an anti-OX40 antibody including thatsequence retains the ability to bind to OX40. In certain embodiments, atotal of 1 to 10 amino acids (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10amino acids) have been substituted, inserted, and/or deleted in SEQ IDNO: 16. In certain embodiments, substitutions, insertions, or deletionsoccur in regions outside the HVRs (i.e., in the FRs). Optionally, theanti-OX40 antibodies include the VH sequence in SEQ ID NO: 16, includingpost-translational modifications of that sequence. In a particularembodiment, the VH comprises one, two, or three HVRs selected from: (a)HVR-H1 comprising the amino acid sequence of SEQ ID NO: 2, (b) HVR-H2comprising the amino acid sequence of SEQ ID NO: 3, and (c) HVR-H3comprising the amino acid sequence of SEQ ID NO: 4.

In some instances, the anti-OX40 antibodies that bind to amino acidresidues 266-277 of OX40 may also include a light chain variable domain(VL) having at least 80% (e.g., at least 81%, 82%, 83%, 84%, 85%, 86%,87%, 88%, or 89%), at least 90% (e.g., at least 91%, 92%, 93%, or 94%),or at least 95% (e.g., at least 96%, 97%, 98%, or 99%) sequence identityto, or the sequence of, the amino acid sequence of SEQ ID NO: 17. Incertain embodiments, a VL sequence having at least 80%, 81%, 82%, 83%,84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, or 99% identity contains substitutions (e.g., conservativesubstitutions), insertions, or deletions relative to the referencesequence (SEQ ID NO: 17), but an anti-OX40 antibody including thatsequence retains the ability to bind to OX40. In certain embodiments, atotal of 1 to 10 amino acids (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10amino acids) have been substituted, inserted, and/or deleted in SEQ IDNO: 17. In certain embodiments, the substitutions, insertions, ordeletions occur in regions outside the HVRs (i.e., in the FRs).Optionally, the anti-OX40 antibody comprises the VL sequence in SEQ IDNO: 17, including post-translational modifications of that sequence. Ina particular embodiment, the VL comprises one, two or three HVRsselected from (a) HVR-L1 comprising the amino acid sequence of SEQ IDNO: 9; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 10;and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 11.

In some instances, the anti-OX40 antibodies that bind to amino acidresidues 266-277 of OX40 include both VH and VL sequences having atleast 80% (e.g., at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, or89%), at least 90% (e.g., at least 91%, 92%, 93%, or 94%), or at least95% (e.g., at least 96%, 97%, 98%, or 99%) sequence identity to, or thesequences of, the amino acid sequences of SEQ ID NOs: 16 and 17,respectively, and may or may not include post-translationalmodifications of those sequences.

In other instances, the invention provides antibodies that specificallybind OX40, wherein the antibodies include (a) an HVR-H1 comprising theamino acid sequence of SEQ ID NO: 2; (b) an HVR-H2 comprising the aminoacid sequence of SEQ ID NO: 3; (c) an HVR-H3 comprising the amino acidsequence of SEQ ID NO: 4; (d) an HVR-L1 comprising the amino acidsequence of SEQ ID NO: 9; (e) an HVR-L2 comprising the amino acidsequence of SEQ ID NO: 10; and (f) an HVR-L3 comprising the amino acidsequence of SEQ ID NO: 11. In some instances, these anti-OX40 antibodiesinclude the following FRs: (a) FR-H1 comprising the amino acid sequenceof SEQ ID NO: 5; (b) FR-H2 comprising the amino acid sequence of SEQ IDNO: 6; (c) FR-H3 comprising the amino acid sequence of SEQ ID NO: 7; and(d) FR-H4 comprising the amino acid sequence of SEQ ID NO: 8 and mayadditionally or alternatively include (e) FR-L1 comprising the aminoacid sequence of SEQ ID NO: 12; (f) FR-L2 comprising the amino acidsequence of SEQ ID NO: 13; (g) FR-L3 comprising the amino acid sequenceof SEQ ID NO: 14; and (h) FR-L4 comprising the amino acid sequence ofSEQ ID NO: 15. In some embodiments, for example, the anti-OX40antibodies include both a VH and a VL sequence including the sequencesof the amino acid sequences of SEQ ID NOs: 16 and 17, respectively, andmay or may not include post-translational modifications.

For example, the invention features anti-OX40 antibodies, such as theanti-OX40 antibody SP197, with the following heavy and light chainvariable region sequences.

The amino acid sequence of the heavy chain variable region is thefollowing (HVR sequences underlined):

(SEQ ID NO: 16) QSLEESGGRLVAPGGSLTLTCTVSGIDLSSDNIQWVRQAPGKGLEWIGAVDYNNKPFYANWAKGRFTISKTSSTTVDLKMTSL TTEDTATYFCAKNTFSPWGPGTLVTVSS

The amino acid sequence of the light chain variable region is thefollowing (HVR sequences underlined):

(SEQ ID NO: 17) DPAMTQTPSSTSAAVGGTVTINCQSSQSVYNANHLSWFQQKPGQPPKRLIYYISTPDSGVPPRFSGSGSGTQFTLTISGVQCDDA ATYYCAALNSDEVFTFGGGTEVVVK

In some instances, anti-OX40 antibodies of the invention are antibodiesthat compete for binding to OX40 with any one or more of the anti-OX40antibodies described above. In some instances, anti-OX40 antibodies ofthe invention are antibodies that bind to the same epitope orsubstantially the same epitope as any one or more of the anti-OX40antibodies described above.

In some instances, an anti-OX40 antibody according to any of the aboveembodiments may be a monoclonal antibody, comprising a chimeric,humanized, or human antibody. In one embodiment, an anti-OX40 antibodyis an antibody fragment, for example, a Fv, Fab, Fab′, scFv, diabody, orF(ab′)₂ fragment. In another embodiment, the antibody is a full-lengthantibody, e.g., an intact IgG antibody (e.g., an intact IgG1 antibody)or other antibody class or isotype as defined herein.

It should be understood that the anti-OX40 antibodies of the invention,although useful for the detection of the presence or the expressionlevel of OX40 in a biological sample as exemplified by the Examplesbelow, may also be used or adapted for therapeutic use.

In further aspects, the anti-OX40 antibodies according to any of theabove embodiments may incorporate any of the features, singly or incombination, as described in Sections 1-5 below.

1. Antibody Affinity

In certain embodiments, an antibody provided herein has a dissociationconstant (Kd) of ≤1 μM, ≤100 nM, ≤10 nM, ≤1 nM, ≤0.1 nM, ≤0.01 nM, or≤0.001 nM (e.g. 10⁻⁸ M or less, e.g. from 10⁻⁸ M to 10⁻¹³ M, e.g., from10⁻⁹ M to 10⁻¹³ M).

In one embodiment, Kd is measured by a radiolabeled antigen bindingassay (RIA) performed with the Fab version of an antibody of interestand its antigen as described by the following assay. Solution bindingaffinity of Fabs for antigen is measured by equilibrating Fab with aminimal concentration of (¹²⁵I)-labeled antigen in the presence of atitration series of unlabeled antigen, then capturing bound antigen withan anti-Fab antibody-coated plate (see, e.g., Chen et al. J. Mol. Biol.293: 865-881, 1999). To establish conditions for the assay, MICROTITER®multi-well plates (Thermo Scientific) are coated overnight with 5 μg/mlof a capturing anti-Fab antibody (Cappel Labs) in 50 mM sodium carbonate(pH 9.6), and subsequently blocked with 2% (w/v) bovine serum albumin inPBS for two to five hours at room temperature (approximately 23° C.). Ina non-adsorbent plate (Nunc #269620), 100 pM or 26 pM [¹²⁵I]-antigen aremixed with serial dilutions of a Fab of interest (e.g., consistent withassessment of the anti-VEGF antibody, Fab-12, in Presta et al. CancerRes. 57: 4593-4599, 1997). The Fab of interest is then incubatedovernight; however, the incubation may continue for a longer period(e.g., about 65 hours) to ensure that equilibrium is reached.Thereafter, the mixtures are transferred to the capture plate forincubation at room temperature (e.g., for one hour). The solution isthen removed and the plate washed eight times with 0.1% polysorbate 20(TWEEN-20™) in PBS. When the plates have dried, 150 μl/well ofscintillant (MICROSCINT-20™; Packard) is added, and the plates arecounted on a TOPCOUNT™ gamma counter (Packard) for ten minutes.Concentrations of each Fab that give less than or equal to 20% ofmaximal binding are chosen for use in competitive binding assays.

According to another embodiment, Kd is measured using surface plasmonresonance assays using a BIACORE®-2000 or a BIACORE®-3000 (BIAcore,Inc., Piscataway, N.J.) at 25° C. with immobilized antigen CM5 chips at˜10 response units (RU). Briefly, carboxymethylated dextran biosensorchips (CM5, BIACORE, Inc.) are activated withN-ethyl-N′-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) andN-hydroxysuccinimide (NHS) according to the supplier's instructions.Antigen is diluted with 10 mM sodium acetate, pH 4.8, to 5 μg/ml (−0.2μM) before injection at a flow rate of 5 μl/minute to achieveapproximately 10 response units (RU) of coupled protein. Following theinjection of antigen, 1 M ethanolamine is injected to block unreactedgroups. For kinetics measurements, two-fold serial dilutions of Fab(0.78 nM to 500 nM) are injected in PBS with 0.05% polysorbate 20(TWEEN-20™) surfactant (PBST) at 25° C. at a flow rate of approximately25 μl/min Association rates (k_(on)) and dissociation rates (k_(off))are calculated using a simple one-to-one Langmuir binding model(BIACORE® Evaluation Software version 3.2) by simultaneously fitting theassociation and dissociation sensorgrams. The equilibrium dissociationconstant (Kd) is calculated as the ratio k_(off)/k_(on). See, e.g., Chenet al. J. Mol. Biol. 293: 865-881, 1999. If the on-rate exceeds 10⁶ M⁻¹s⁻¹ by the surface plasmon resonance assay above, then the on-rate canbe determined by using a fluorescent quenching technique that measuresthe increase or decrease in fluorescence emission intensity(excitation=295 nm; emission=340 nm, 16 nm band-pass) at 25° C. of a 20nM anti-antigen antibody (Fab form) in PBS, pH 7.2, in the presence ofincreasing concentrations of antigen as measured in a spectrometer, suchas a stop-flow equipped spectrophometer (Aviv Instruments) or a8000-series SLM-AMINCO™ spectrophotometer (ThermoSpectronic) with astirred cuvette.

2. Antibody Fragments

In certain embodiments, an antibody provided herein is an antibodyfragment. Antibody fragments include, but are not limited to, Fab, Fab′,Fab′-SH, F(ab′)2, Fv, and scFv fragments, and other fragments describedbelow. For a review of certain antibody fragments, see Hudson et al.Nat. Med. 9: 129-134, 2003. For a review of scFv fragments, see, e.g.,Pluckthun. The Pharmacology of Monoclonal Antibodies. Vol. 113, pp.269-315, Rosenburg and Moore eds. Springer-Verlag, New York, 1994; seealso WO 93/16185; and U.S. Pat. Nos. 5,571,894 and 5,587,458. Fordiscussion of Fab and F(ab′)2 fragments comprising salvage receptorbinding epitope residues and having increased in vivo half-life, seeU.S. Pat. No. 5,869,046.

Diabodies are antibody fragments with two antigen-binding sites that maybe bivalent or bispecific. See, for example, EP 404,097; WO 1993/01161;Hudson et al. Nat. Med. 9: 129-134, 2003; and Hollinger et al. Proc.Natl. Acad. Sci. USA. 90: 6444-6448, 1993. Triabodies and tetrabodiesare also described in Hudson et al. Nat. Med. 9:129-134, 2003.

Single-domain antibodies are antibody fragments comprising all or aportion of the heavy chain variable domain or all or a portion of thelight chain variable domain of an antibody. In certain embodiments, asingle-domain antibody is a human single-domain antibody (Domantis,Inc., Waltham, Mass.; see, e.g., U.S. Pat. No. 6,248,516).

Antibody fragments can be made by various techniques, including but notlimited to proteolytic digestion of an intact antibody as well asproduction by recombinant host cells (e.g. E. coli or phage), asdescribed herein.

3. Chimeric and Humanized Antibodies

In certain embodiments, an antibody provided herein is a chimericantibody.

Certain chimeric antibodies are described, e.g., in U.S. Pat. No.4,816,567; and Morrison et al. Proc. Natl. Acad. Sci. USA. 81:6851-6855, 1984. In one example, a chimeric antibody comprises anon-human variable region (e.g., a variable region derived from a mouse,rat, hamster, rabbit, or non-human primate, such as a monkey) and ahuman constant region. In a further example, a chimeric antibody is a“class switched” antibody in which the class or subclass has beenchanged from that of the parent antibody. Chimeric antibodies includeantigen-binding fragments thereof.

In certain embodiments, a chimeric antibody is a humanized antibody.Typically, a non-human antibody is humanized to reduce immunogenicity tohumans, while retaining the specificity and affinity of the parentalnon-human antibody. Generally, a humanized antibody comprises one ormore variable domains in which HVRs, e.g., CDRs, (or portions thereof)are derived from a non-human antibody, and 1-Rs (or portions thereof)are derived from human antibody sequences. A humanized antibodyoptionally will also comprise at least a portion of a human constantregion. In some embodiments, some FR residues in a humanized antibodyare substituted with corresponding residues from a non-human antibody(e.g., the antibody from which the HVR residues are derived), e.g., torestore or improve antibody specificity or affinity.

Humanized antibodies and methods of making them are reviewed, e.g., inAlmagro et al. Front. Biosci. 13: 1619-1633, 2008, and are furtherdescribed, e.g., in Riechmann et al. Nature. 332: 323-329, 1988; Queenet al. Proc. Natl. Acad. Sci. USA. 86: 10029-10033, 1989; U.S. Pat. Nos.5,821,337, 7,527,791, 6,982,321, and 7,087,409; Kashmiri et al. Methods.36: 25-34, 2005 (describing SDR (a-CDR) grafting); Padlan. Mol. Immunol.28: 489-498, 1991 (describing “resurfacing”); DaU'Acqua et al. Methods.36: 43-60, 2005 (describing “FR shuffling”); and Osbourn et al. Methods36: 61-68, 2005 and Klimka et al. Br. J. Cancer. 83: 252-260, 2000(describing the “guided selection” approach to FR shuffling).

Human framework regions that may be used for humanization include butare not limited to: framework regions selected using the “best-fit”method (see, e.g., Sims et al. J. Immunol. 151: 2296, 1993); frameworkregions derived from the consensus sequence of human antibodies of aparticular subgroup of light or heavy chain variable regions (see, e.g.,Carter et al. Proc. Natl. Acad. Sci. USA. 89: 4285, 1992; and Presta etal. J. Immunol. 151: 2623, 1993); human mature (somatically mutated)framework regions or human germline framework regions (see, e.g.,Almagro et al. Front. Biosci. 13: 1619-1633, 2008); and frameworkregions derived from screening FR libraries (see, e.g., Baca et al. J.Biol. Chem. 272: 10678-10684, 1997 and Rosok et al. J. Biol. Chem. 271:22611-22618, 1996).

4. Multispecific Antibodies

In certain embodiments, an antibody provided herein is a multispecificantibody, e.g., a bispecific antibody. Multispecific antibodies aremonoclonal antibodies that have binding specificities for at least twodifferent sites. In certain embodiments, one of the bindingspecificities is for OX40 and the other is for any other antigen. Incertain embodiments, bispecific antibodies may bind to two differentepitopes of OX40. Bispecific antibodies may also be used to localizecytotoxic agents to cells which express OX40. Bispecific antibodies canbe prepared as full-length antibodies or antibody fragments.

Techniques for making multispecific antibodies include, but are notlimited to, recombinant co-expression of two immunoglobulin heavychain-light chain pairs having different specificities (see Milstein etal. Nature. 305: 537, 1983, WO 93/08829, and Traunecker et al. EMBO J.10: 3655, 1991), and “knob-in-hole” engineering (see, e.g., U.S. Pat.No. 5,731,168). Multi-specific antibodies may also be made byengineering electrostatic steering effects for making antibodyFc-heterodimeric molecules (WO 2009/089004A1); cross-linking two or moreantibodies or fragments (see, e.g., U.S. Pat. No. 4,676,980, and Brennanet al. Science. 229: 81, 1985); using leucine zippers to producebi-specific antibodies (see, e.g., Kostelny et al. J. Immunol. 148(5):1547-1553, 1992); using “diabody” technology for making bispecificantibody fragments (see, e.g., Hollinger et al. Proc. Natl. Acad. Sci.USA., 90: 6444-6448, 1993); and using single-chain Fv (sFv) dimers (see,e.g. Gruber et al. J. Immunol. 152: 5368, 1994); and preparingtrispecific antibodies as described, e.g., in Tutt et al. J. Immunol.147: 60, 1991. Engineered antibodies with three or more functionalantigen binding sites, including “Octopus antibodies,” are also includedherein (see, e.g. US 2006/0025576A1).

The antibody or fragment herein also includes a “Dual Acting FAb” or“DAF” comprising an antigen binding site that binds to OX40 as well asanother, different antigen (see, e.g., US 2008/0069820).

5. Antibody Variants

In certain embodiments, amino acid sequence variants of the antibodiesprovided herein are contemplated. For example, it may be desirable toimprove the binding affinity and/or other biological properties of theantibody Amino acid sequence variants of an antibody may be prepared byintroducing appropriate modifications into the nucleotide sequenceencoding the antibody, or by peptide synthesis. Such modificationsinclude, for example, deletions from, and/or insertions into and/orsubstitutions of residues within the amino acid sequences of theantibody. Any combination of deletion, insertion, and substitution canbe made to arrive at the final construct, provided that the finalconstruct possesses the desired characteristics, e.g., antigen-binding.

a) Substitution, Insertion, and Deletion Variants

In certain embodiments, antibody variants having one or more amino acidsubstitutions are provided. Sites of interest for substitutionalmutagenesis include the HVRs and FRs. Conservative substitutions areshown in Table 1 under the heading of “preferred substitutions.” Moresubstantial changes are provided in Table 1 under the heading of“exemplary substitutions,” and as further described below in referenceto amino acid side chain classes. Amino acid substitutions may beintroduced into an antibody of interest and the products screened for adesired activity, e.g., retained/improved antigen binding, decreasedimmunogenicity, or improved ADCC or CDC.

TABLE 1 Exemplary and Preferred Amino Acid Substitutions OriginalExemplary Preferred Residue Substitutions Substitutions Ala (A) Val;Leu; Ile Val Arg (R) Lys; Gln; Asn Lys Asn (N) Gln; His; Asp, Lys; ArgGln Asp (D) Glu; Asn Glu Cys (C) Ser; Ala Ser Gln (Q) Asn; Glu Asn Glu(E) Asp; Gln Asp Gly (G) Ala Ala His (H) Asn; Gln; Lys; Arg Arg Ile (I)Leu; Val; Met; Ala; Phe; Leu Norleucine Leu (L) Norleucine; Ile; Val;Met; Ala; Phe Ile Lys (K) Arg; Gln; Asn Arg Met (M) Leu; Phe; Ile LeuPhe (F) Trp; Leu; Val; Ile; Ala; Tyr Tyr Pro (P) Ala Ala Ser (S) Thr ThrThr (T) Val; Ser Ser Trp (W) Tyr; Phe Tyr Tyr (Y) Trp; Phe; Thr; Ser PheVal (V) Ile; Leu; Met; Phe; Ala; Leu Norleucine

Amino acids may be grouped according to common side-chain properties:

-   -   (1) hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile;    -   (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln;    -   (3) acidic: Asp, Glu;    -   (4) basic: His, Lys, Arg;    -   (5) residues that influence chain orientation: Gly, Pro;    -   (6) aromatic: Trp, Tyr, Phe.

Non-conservative substitutions will entail exchanging a member of one ofthese classes for another class.

One type of substitutional variant involves substituting one or morehypervariable region residues of a parent antibody (e.g. a humanized orhuman antibody). Generally, the resulting variant(s) selected forfurther study will have modifications (e.g., improvements) in certainbiological properties (e.g., increased affinity, reduced immunogenicity)relative to the parent antibody and/or will have substantially retainedcertain biological properties of the parent antibody. An exemplarysubstitutional variant is an affinity matured antibody, which may beconveniently generated, e.g., using phage display-based affinitymaturation techniques such as those described herein. Briefly, one ormore HVR residues are mutated and the variant antibodies displayed onphage and screened for a particular biological activity (e.g. bindingaffinity).

Alterations (e.g., substitutions) may be made in HVRs, e.g., to improveantibody affinity. Such alterations may be made in HVR “hotspots,” i.e.,residues encoded by codons that undergo mutation at high frequencyduring the somatic maturation process (see, e.g., Chowdhury. MethodsMol. Biol. 207: 179-196, 2008), and/or SDRs (a-CDRs), with the resultingvariant VH or VL being tested for binding affinity. Affinity maturationby constructing and reselecting from secondary libraries has beendescribed, e.g., in Hoogenboom et al. Methods in Molecular Biology. 178:1-37, O'Brien et al. eds., Human Press, Totowa, N.J., 2001. In someembodiments of affinity maturation, diversity is introduced into thevariable genes chosen for maturation by any of a variety of methods(e.g., error-prone PCR, chain shuffling, or oligonucleotide-directedmutagenesis). A secondary library is then created. The library is thenscreened to identify any antibody variants with the desired affinity.Another method to introduce diversity involves HVR-directed approaches,in which several HVR residues (e.g., 4-6 residues at a time) arerandomized HVR residues involved in antigen binding may be specificallyidentified, e.g., using alanine scanning mutagenesis or modeling. HVR-H3and HVR-L3 in particular are often targeted.

In certain embodiments, substitutions, insertions, or deletions mayoccur within one or more HVRs so long as such alterations do notsubstantially reduce the ability of the antibody to bind antigen. Forexample, conservative alterations (e.g., conservative substitutions asprovided herein) that do not substantially reduce binding affinity maybe made in HVRs. Such alterations may be outside of HVR “hotspots” orSDRs. In certain embodiments of the variant VH and VL sequences providedabove, each HVR either is unaltered, or contains no more than one, twoor three amino acid substitutions.

A useful method for identification of residues or regions of an antibodythat may be targeted for mutagenesis is called “alanine scanningmutagenesis” as described by Cunningham et al. Science. 244: 1081-1085,1989. In this method, a residue or group of target residues (e.g.,charged residues such as Arg, Asp, His, Lys, and Glu) are identified andreplaced by a neutral or negatively charged amino acid (e.g., alanine orpolyalanine) to determine whether the interaction of the antibody withantigen is affected. Further substitutions may be introduced at theamino acid locations demonstrating functional sensitivity to the initialsubstitutions. Alternatively, or additionally, a crystal structure of anantigen-antibody complex to identify contact points between the antibodyand antigen. Such contact residues and neighboring residues may betargeted or eliminated as candidates for substitution. Variants may bescreened to determine whether they contain the desired properties.

Amino acid sequence insertions include amino- and/or carboxyl-terminalfusions ranging in length from one residue to polypeptides containing ahundred or more residues, as well as intrasequence insertions of singleor multiple amino acid residues. Examples of terminal insertions includean antibody with an N-terminal methionyl residue. Other insertionalvariants of the antibody molecule include the fusion to the N- orC-terminus of the antibody to an enzyme (e.g. for ADEPT) or apolypeptide which increases the serum half-life of the antibody.

b) Glycosylation Variants

In certain embodiments, an antibody provided herein is altered toincrease or decrease the extent to which the antibody is glycosylated.Addition or deletion of glycosylation sites to an antibody may beconveniently accomplished by altering the amino acid sequence such thatone or more glycosylation sites is created or removed.

Where the antibody comprises an Fc region, the carbohydrate attachedthereto may be altered. Native antibodies produced by mammalian cellstypically comprise a branched, biantennary oligosaccharide that isgenerally attached by an N-linkage to Asn297 of the CH2 domain of the Fcregion. See, e.g., Wright et al. TIBTECH. 15: 26-32, 1997. Theoligosaccharide may include various carbohydrates, e.g., mannose,N-acetyl glucosamine (GlcNAc), galactose, and sialic acid, as well as afucose attached to a GlcNAc in the “stem” of the biantennaryoligosaccharide structure. In some embodiments, modifications of theoligosaccharide in an antibody of the invention may be made in order tocreate antibody variants with certain improved properties.

In one embodiment, antibody variants are provided having a carbohydratestructure that lacks fucose attached (directly or indirectly) to an Fcregion. For example, the amount of fucose in such antibody may be from1% to 80%, from 1% to 65%, from 5% to 65%, or from 20% to 40%. Theamount of fucose is determined by calculating the average amount offucose within the sugar chain at Asn297, relative to the sum of allglycostructures attached to Asn297 (e.g., complex, hybrid and highmannose structures) as measured by MALDI-TOF mass spectrometry, asdescribed in WO 2008/077546, for example. Asn297 refers to theasparagine residue located at about position 297 in the Fc region (EUnumbering of Fc region residues); however, Asn297 may also be locatedabout ±3 amino acids upstream or downstream of position 297, i.e.,between positions 294 and 300, due to minor sequence variations inantibodies. Such fucosylation variants may have improved ADCC function.See, e.g., US Patent Publication Nos. US 2003/0157108 and US2004/0093621. Examples of publications related to “defucosylated” or“fucose-deficient” antibody variants include: US 2003/0157108; WO2000/61739; WO 2001/29246; US 2003/0115614; US 2002/0164328; US2004/0093621; US 2004/0132140; US 2004/0110704; US 2004/0110282; US2004/0109865; WO 2003/085119; WO 2003/084570; WO 2005/035586; WO2005/035778; WO2005/053742; WO2002/031140; Okazaki et al. J. Mol. Biol.336: 1239-1249, 2004; and Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614,2004. Examples of cell lines capable of producing defucosylatedantibodies include Lec13 CHO cells deficient in protein fucosylation(Ripka et al. Arch. Biochem. Biophys. 249: 533-545, 1986; US2003/0157108; and WO 2004/056312, especially at Example 11), andknockout cell lines, such as alpha-1,6-fucosyltransferase gene, FUT8,knockout CHO cells (see, e.g., Yamane-Ohnuki et al. Biotech. Bioeng. 87:614, 2004; Kanda et al. Biotechnol. Bioeng. 94(4): 680-688, 2006; andWO2003/085107).

Antibodies variants are further provided with bisected oligosaccharides,e.g., in which a biantennary oligosaccharide attached to the Fc regionof the antibody is bisected by GlcNAc. Such antibody variants may havereduced fucosylation and/or improved ADCC function. Examples of suchantibody variants are described, e.g., in WO 2003/011878; U.S. Pat. No.6,602,684; and US 2005/0123546. Antibody variants with at least onegalactose residue in the oligosaccharide attached to the Fc region arealso provided. Such antibody variants may have improved CDC function.Such antibody variants are described, e.g., in WO 1997/30087; WO1998/58964; and WO 1999/22764.

c) Fc Region Variants

In certain embodiments, one or more amino acid modifications may beintroduced into the Fc region of an anti-OX40 antibody of the invention(e.g., SP197) provided herein, thereby generating an Fc region variant.The Fc region variant may comprise a human Fc region sequence (e.g., ahuman IgG₁, IgG₂, IgG₃ or IgG₄ Fc region) comprising an amino acidmodification (e.g., a substitution) at one or more amino acid positions.

In certain embodiments, the invention contemplates an antibody variantthat possesses some but not all effector functions, which make it adesirable candidate for applications in which the half life of theantibody in vivo is important yet certain effector functions (such ascomplement and ADCC) are unnecessary or deleterious. In vitro and/or invivo cytotoxicity assays can be conducted to confirm thereduction/depletion of CDC and/or ADCC activities. For example, Fcreceptor (FcR) binding assays can be conducted to ensure that theantibody lacks FcyR binding (hence likely lacking ADCC activity), butretains FcRn binding ability. The primary cells for mediating ADCC, NKcells, express FcyRIII only, whereas monocytes express FcyRI, FcyRII andFcyRIII. FcR expression on hematopoietic cells is summarized in Table 3on page 464 of Ravetch et al. Annu. Rev. Immunol. 9: 457-492, 1991.Non-limiting examples of in vitro assays to assess ADCC activity of amolecule of interest is described in U.S. Pat. Nos. 5,500,362 and5,821,337; Hellstrom et al. Proc. Natl. Acad. Sci. USA. 83: 7059-7063,1986; Hellstrom et al. Proc. Natl Acad. Sci. USA. 82: 1499-1502, 1985;and Bruggemann et al. J. Exp. Med. 166: 1351-1361, 1987. Alternatively,non-radioactive assays methods may be employed (see, for example, ACTI™non-radioactive cytotoxicity assay for flow cytometry (CellTechnology,Inc. Mountain View, Calif.; and CytoTox 96® non-radioactive cytotoxicityassay (Promega, Madison, Wis.). Useful effector cells for such assaysinclude peripheral blood mononuclear cells (PBMC) and Natural Killer(NK) cells. Alternatively, or additionally, ADCC activity of themolecule of interest may be assessed in vivo, e.g., in an animal modelsuch as that disclosed in Clynes et al. Proc. Natl. Acad. Sci. USA.95:652-656, 1998. C1q binding assays may also be carried out to confirmthat the antibody is unable to bind C1q and hence lacks CDC activity.See, e.g., C1q and C3c binding ELISA in WO 2006/029879 and WO2005/100402. To assess complement activation, a CDC assay may beperformed (see, e.g., Gazzano-Santoro et al. J. Immunol. Methods. 202:163, 1996; Cragg et al. Blood. 101: 1045-1052, 2003; and Cragg et al.Blood 103: 2738-2743, 2004. FcRn binding and in vivo clearance/half lifedeterminations can also be performed using methods known in the art(see, e.g., Petkova et al. Intl. Immunol. 18(12): 1759-1769, 2006).

Antibodies with reduced effector function include those withsubstitution of one or more of Fc region residues 238, 265, 269, 270,297, 327, and 329 (U.S. Pat. No. 6,737,056). Such Fc mutants include Fcmutants with substitutions at two or more of amino acid positions 265,269, 270, 297, and 327, including the so-called “DANA” Fc mutant withsubstitution of residues 265 and 297 to alanine (U.S. Pat. No.7,332,581).

Certain antibody variants with improved or diminished binding to FcRsare described. See, e.g., U.S. Pat. No. 6,737,056; WO 2004/056312; andShields et al. J. Biol. Chem. 9(2): 6591-6604, 2001.

In certain embodiments, an antibody variant comprises an Fc region withone or more amino acid substitutions which improve ADCC, e.g.,substitutions at positions 298, 333, and/or 334 of the Fc region (EUnumbering of residues).

In some embodiments, alterations are made in the Fc region that resultin altered (i.e., either improved or diminished) C1q binding and/orComplement Dependent Cytotoxicity (CDC), e.g., as described in U.S. Pat.No. 6,194,551, WO 99/51642, and Idusogie et al. J. Immunol. 164:4178-4184, 2000.

Antibodies with increased half lives and improved binding to theneonatal Fc receptor (FcRn), which is responsible for the transfer ofmaternal IgGs to the fetus (Guyer et al. J. Immunol. 117: 587, 1976 andKim et al, J. Immunol. 24: 249, 1994), are described in US PatentApplication No. 2005/0014934. Those antibodies comprise an Fc regionwith one or more substitutions therein which improve binding of the Fcregion to FcRn. Such Fc variants include those with substitutions at oneor more of Fc region residues: 238, 256, 265, 272, 286, 303, 305, 307,311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, 413, 424, or 434,e.g., substitution of Fc region residue 434 (U.S. Pat. No. 7,371,826).See also Duncan et al. Nature. 322:738-740, 1988; U.S. Pat. Nos.5,648,260 and 5,624,821; and WO 94/29351 concerning other examples of Fcregion variants.

d) Cysteine Engineered Antibody Variants

In certain embodiments, it may be desirable to create cysteineengineered antibodies, e.g., “thioMAbs,” in which one or more residuesof an antibody are substituted with cysteine residues. In particularembodiments, the substituted residues occur at accessible sites of theantibody. By substituting those residues with cysteine, reactive thiolgroups are thereby positioned at accessible sites of the antibody andmay be used to conjugate the antibody to other moieties, such as drugmoieties or linker-drug moieties, to create an immunoconjugate, asdescribed further herein. In certain embodiments, any one or more of thefollowing residues may be substituted with cysteine: V205 (Kabatnumbering) of the light chain; A118 (EU numbering) of the heavy chain;and 5400 (EU numbering) of the heavy chain Fc region. Cysteineengineered antibodies may be generated as described, e.g., in U.S. Pat.No. 7,521,541.

e) Antibody Derivatives

In certain embodiments, an anti-OX40 antibody of the invention (e.g.,SP197) provided herein may be further modified to contain additionalnonproteinaceous moieties that are known in the art and readilyavailable. The moieties suitable for derivatization of the antibodyinclude but are not limited to water soluble polymers. Non-limitingexamples of water soluble polymers include, but are not limited to,polyethylene glycol (PEG), copolymers of ethylene glycol/propyleneglycol, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene/maleicanhydride copolymer, polyaminoacids (either homopolymers or randomcopolymers), and dextran or poly(n-vinyl pyrrolidone)polyethyleneglycol, propropylene glycol homopolymers, prolypropylene oxide/ethyleneoxide co-polymers, polyoxyethylated polyols (e.g., glycerol), polyvinylalcohol, and mixtures thereof. Polyethylene glycol propionaldehyde mayhave advantages in manufacturing due to its stability in water. Thepolymer may be of any molecular weight, and may be branched orunbranched. The number of polymers attached to the antibody may vary,and if more than one polymer is attached, they can be the same ordifferent molecules. In general, the number and/or type of polymers usedfor derivatization can be determined based on considerations including,but not limited to, the particular properties or functions of theantibody to be improved, whether the antibody derivative will be used ina therapy under defined conditions, etc.

In another embodiment, conjugates of an antibody and nonproteinaceousmoiety that may be selectively heated by exposure to radiation areprovided. In one embodiment, the nonproteinaceous moiety is a carbonnanotube (Kam et al. Proc. Natl. Acad. Sci. USA. 102: 11600-11605,2005). The radiation may be of any wavelength, and includes, but is notlimited to, wavelengths that do not harm ordinary cells, but which heatthe nonproteinaceous moiety to a temperature at which cells proximal tothe antibody-nonproteinaceous moiety are killed.

B. Recombinant Methods and Compositions

Antibodies may be produced using recombinant methods and compositions,e.g., as described in U.S. Pat. No. 4,816,567. In one embodiment,isolated nucleic acid encoding an anti-OX40 antibody described herein(e.g., SP197) is provided. Such nucleic acid may encode an amino acidsequence comprising the VL and/or an amino acid sequence comprising theVH of the antibody (e.g., the light and/or heavy chains of theantibody). In a further embodiment, one or more vectors (e.g.,expression vectors) comprising such nucleic acid are provided. In afurther embodiment, a host cell comprising such nucleic acid isprovided. In one such embodiment, a host cell comprises (e.g., has beentransformed with): (1) a vector comprising a nucleic acid that encodesan amino acid sequence comprising the VL of the antibody and an aminoacid sequence comprising the VH of the antibody, or (2) a first vectorcomprising a nucleic acid that encodes an amino acid sequence comprisingthe VL of the antibody and a second vector comprising a nucleic acidthat encodes an amino acid sequence comprising the VH of the antibody.In one embodiment, the host cell is eukaryotic, e.g. a Chinese HamsterOvary (CHO) cell or lymphoid cell (e.g., Y0, NSO, Sp20 cell). In oneembodiment, a method of making an anti-OX40 antibody is provided,wherein the method comprises culturing a host cell comprising a nucleicacid encoding the antibody, as provided above, under conditions suitablefor expression of the antibody, and optionally recovering the antibodyfrom the host cell (or host cell culture medium).

For recombinant production of an anti-OX40 antibody (e.g., SP197),nucleic acid encoding an antibody, e.g., as described above, is isolatedand inserted into one or more vectors for further cloning and/orexpression in a host cell. Such nucleic acid may be readily isolated andsequenced using conventional procedures (e.g., by using oligonucleotideprobes that are capable of binding specifically to genes encoding theheavy and light chains of the antibody).

Suitable host cells for cloning or expression of antibody-encodingvectors include prokaryotic or eukaryotic cells described herein. Forexample, antibodies may be produced in bacteria, in particular whenglycosylation and Fc effector function are not needed. For expression ofantibody fragments and polypeptides in bacteria, see, e.g., U.S. Pat.Nos. 5,648,237, 5,789,199, and 5,840,523. See also Charlton. Methods inMolecular Biology. Vol. 248, pp. 245-254, B. K. C. Lo, ed., HumanaPress, Totowa, N.J., 2003, describing expression of antibody fragmentsin E. coli. After expression, the antibody may be isolated from thebacterial cell paste in a soluble fraction and can be further purified.

In addition to prokaryotes, eukaryotic microbes such as filamentousfungi or yeast are suitable cloning or expression hosts forantibody-encoding vectors, including fungi and yeast strains whoseglycosylation pathways have been “humanized,” resulting in theproduction of an antibody with a partially or fully human glycosylationpattern. See Gerngross. Nat. Biotech. 22: 1409-1414, 2004 and Li et al.Nat. Biotech. 24: 210-215, 2006.

Suitable host cells for the expression of glycosylated antibody are alsoderived from multicellular organisms (invertebrates and vertebrates).Examples of invertebrate cells include plant and insect cells. Numerousbaculoviral strains have been identified which may be used inconjunction with insect cells, particularly for transfection ofSpodoptera frugiperda cells.

Plant cell cultures can also be utilized as hosts. See, e.g., U.S. Pat.Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429(describing PLANTIBODIES™ technology for producing antibodies intransgenic plants).

Vertebrate cells may also be used as hosts. For example, mammalian celllines that are adapted to grow in suspension may be useful. Otherexamples of useful mammalian host cell lines are monkey kidney CV1 linetransformed by SV40 (COS-7); human embryonic kidney line (293 or 293cells as described, e.g., in Graham et al. J. Gen Virol. 36:59, 1977);baby hamster kidney cells (BHK); mouse Sertoli cells (TM4 cells asdescribed, e.g., in Mather. Biol. Reprod. 23:243-251, 1980); monkeykidney cells (CV1); African green monkey kidney cells (VERO-76); humancervical carcinoma cells (HELA); canine kidney cells (MDCK; buffalo ratliver cells (BRL 3 A); human lung cells (W138); human liver cells (HepG2); mouse mammary tumor (MMT 060562); TRI cells, as described, e.g., inMather et al. Annals N.Y. Acad. Sci. 383:44-68, 1982; MRC 5 cells; andFS4 cells. Other useful mammalian host cell lines include Chinesehamster ovary (CHO) cells, including DHFR″ CHO cells (Urlaub et al.Proc. Natl. Acad. Sci. USA. 77: 4216, 1980); and myeloma cell lines suchas Y0, NS0 and Sp2/0. For a review of certain mammalian host cell linessuitable for antibody production, see, e.g., Yazaki et al. Methods inMolecular Biology. Vol. 248, pp. 255-268, B. K. C. Lo, ed., HumanaPress, Totowa, N.J., 2003.

C. Assays

Anti-OX40 antibodies provided herein may be identified, screened for, orcharacterized for their physical/chemical properties and/or biologicalactivities by various assays known in the art.

1. Binding Assays and Other Assays

In one aspect, an antibody of the invention is tested for its antigenbinding activity, e.g., by known methods such as ELISA, Western blot,immunohistochemistry, immunofluorescence, etc.

In another aspect, competition assays may be used to identify anantibody that competes with any one of the antibodies of the inventionfor binding to OX40 (e.g., anti-OX40 antibody SP197). In certainembodiments, such a competing antibody binds to the same epitope (e.g.,a linear or a conformational epitope) that is bound by any one of theantibodies of the invention (e.g., anti-OX40 antibody SP197). Detailedexemplary methods for mapping an epitope to which an antibody binds areprovided in Morris “Epitope Mapping Protocols,” in Methods in MolecularBiology Vol. 66 (Humana Press, Totowa, N.J., 1996).

In an exemplary competition assay, immobilized OX40 is incubated in asolution comprising a first labeled antibody that binds to OX40 (e.g.,anti-OX40 antibody SP197) and a second unlabeled antibody that is beingtested for its ability to compete with the first antibody for binding toOX40. The second antibody may be present in a hybridoma supernatant. Asa control, immobilized OX40 is incubated in a solution comprising thefirst labeled antibody but not the second unlabeled antibody. Afterincubation under conditions permissive for binding of the first antibodyto OX40, excess unbound antibody is removed, and the amount of labelassociated with immobilized OX40 is measured. If the amount of labelassociated with immobilized OX40 is substantially reduced in the testsample relative to the control sample, then that indicates that thesecond antibody is competing with the first antibody for binding toOX40. See, e.g., Harlow et al. Antibodies: A Laboratory Manual. Ch. 14(Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1988).

2. Detection Assays

In one aspect, assays are provided for identifying anti-OX40 antibodiesuseful for detecting the presence of OX40, e.g., in immunohistochemistry(IHC) or immunofluorescence (IF) assays. In certain embodiments, anantibody of the invention is tested for such activity.

D. Immunoconjugates

The invention also provides immunoconjugates comprising an anti-OX40antibody herein conjugated to one or more labels and/or agents, such asradioactive isotopes.

In one embodiment, an immunoconjugate comprises an antibody as describedherein conjugated to a radioactive atom to form a radioconjugate. Avariety of radioactive isotopes are available for the production ofradioconjugates. Examples include At²¹¹, I¹³¹, I¹²⁵, Y⁹⁰, Re¹⁸⁶, Re¹⁸⁸,Sm¹⁵³, Bi²¹², P³², Pb²¹² and radioactive isotopes of Lu. When theradioconjugate is used for detection, it may comprise a radioactive atomfor scintigraphic studies, for example tc99m or 1123, or a spin labelfor nuclear magnetic resonance (NMR) imaging (also known as magneticresonance imaging, MRI), such as iodine-123 again, iodine-131,indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17, gadolinium,manganese or iron.

Conjugates of an anti-OX40 antibody and label or agent may be made usinga variety of bifunctional protein coupling agents such asN-succinimidyl-3-(2-pyridyldithio) propionate (SPDP),succinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC),iminothiolane (IT), bifunctional derivatives of imidoesters (such asdimethyl adipimidate HCl), active esters (such as disuccinimidylsuberate), aldehydes (such as glutaraldehyde), bis-azido compounds (suchas bis (p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (suchas bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such astoluene 2,6-diisocyanate), and bis-active fluorine compounds (such as1,5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin canbe prepared as described in Vitetta et al., Science 238:1098 (1987).Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent forconjugation of radionucleotide to the antibody. See WO94/11026. Thelinker may be a “cleavable linker” facilitating release of the label oragent. For example, an acid-labile linker, peptidase-sensitive linker,photolabile linker, dimethyl linker or disulfide-containing linker(Chari et al., Cancer Res. 52:127-131 (1992); U.S. Pat. No. 5,208,020)may be used.

The immunoconjugates herein expressly contemplate, but are not limitedto such conjugates prepared with cross-linker reagents including, butnot limited to, BMPS, EMCS, GMBS, HBVS, LC-SMCC, MBS, MPBH, SBAP, SIA,SIAB, SMCC, SMPB, SMPH, sulfo-EMCS, sulfo-GMBS, sulfo-KMUS, sulfo-MBS,sulfo-SIAB, sulfo-SMCC, and sulfo-SMPB, and SVSB(succinimidyl-(4-vinylsulfone)benzoate) which are commercially available(e.g., from Pierce Biotechnology, Inc., Rockford, Ill., U.S.A.).

E. Methods, Kits, and Compositions for Diagnostics and Detection

In certain embodiments, the anti-OX40 antibodies provided herein (e.g.,SP197) are useful for detecting the presence of OX40 in a biologicalsample. The term “detecting” as used herein encompasses quantitative orqualitative detection.

In one instance, an anti-OX40 antibody (e.g., SP197) for use in a methodof diagnosis or detection is provided. In one instance, for example, amethod of detecting the presence of OX40 in a biological sample,described below, is provided. In certain embodiments, the methodcomprises contacting the biological sample with an anti-OX40 antibody asdescribed herein under conditions permissive for binding of theanti-OX40 antibody to OX40, and detecting whether a complex is formedbetween the anti-OX40 antibody and OX40. Such method may be an in vitroor in vivo method. Anti-OX40 antibodies of the invention (e.g., SP197)can be used, for example, in immunoassays, including, for example,immunohistochemistry (IHC), immunofluorescence (IF), immunoblotting(e.g., Western blotting), flow cytometry, and Enzyme-linkedImmunosorbant Assay (ELISA). In one embodiment, an anti-OX40 antibody isused to select subjects eligible for therapy with a cancerimmunotherapy, for example, where OX40 is a biomarker for selection ofpatients.

In certain instances, labeled anti-OX40 antibodies are provided. Labelsinclude, but are not limited to, labels or moieties that are detecteddirectly (such as fluorescent, chromophoric, electron-dense,chemiluminescent, and radioactive labels), as well as moieties, such asenzymes or ligands, that are detected indirectly, for example, throughan enzymatic reaction or molecular interaction. Exemplary labelsinclude, but are not limited to, the radioisotopes ³²P, ¹⁴C, ¹²⁵I, ³H,and ¹³¹I, fluorophores such as rare earth chelates or fluorescein andits derivatives, rhodamine and its derivatives, dansyl, umbelliferone,luceriferases, e.g., firefly luciferase and bacterial luciferase (U.S.Pat. No. 4,737,456), luciferin, 2,3-dihydrophthalazinediones,horseradish peroxidase (HRP), alkaline phosphatase, β-galactosidase,glucoamylase, lysozyme, saccharide oxidases, e.g., glucose oxidase,galactose oxidase, and glucose-6-phosphate dehydrogenase, heterocyclicoxidases such as uricase and xanthine oxidase, coupled with an enzymethat employs hydrogen peroxide to oxidize a dye precursor such as HRP,lactoperoxidase, or microperoxidase, biotin/avidin, spin labels,bacteriophage labels, stable free radicals, and the like.

In certain instances, the anti-OX40 antibodies provided herein are apart of a kit for immunohistochemically detecting the expression ofhuman OX40 in human tissue samples. In an embodiment, the kits includean antibody as described herein and a set of detection reagents. Thedetection reagents include an antibody capable of binding to theanti-OX40 antibody (termed “secondary antibody”), a detectable entityincluding an enzyme coupled to or adapted to be coupled to the secondaryantibody, and reagents reactive with the enzyme to deposit a chromogenor fluorophore on the sample. In an embodiment, the secondary antibodyhas affinity for immunoglobulins from a specific animal species fromwhich the primary antibody is derived (termed a “species-specificsecondary antibody”). In another embodiment, the secondary antibody isreactive with a tag incorporated into the primary antibody, such as anepitope tag located in the primary amino acid sequence of the primaryantibody or a hapten coupled to a reactive side chain of the primaryantibody. In another embodiment, the enzyme is coupled to the secondaryantibody via a signal amplifier. Signal amplification methods for IHCare known to one of ordinary skill in the art. In some examples, signalamplification includes CAtalyzed Reporter Deposition (CARD), also knownas Tyramide Signal Amplification (TSA™). In one variation of this methodan enzyme-conjugated secondary antibody (such as an HRP-conjugatedsecondary antibody) binds to the primary antibody. Next a substrate ofbiotinylated tyramide (tyramine is 4-(2-aminoethyl)phenol) is used,which presumably becomes a free radical when interacting with the HRPenzyme. The phenolic radical then reacts quickly with the surroundingmaterial, thus depositing or fixing biotin in the vicinity. This processis repeated by providing more substrate (biotinylated tyramide) andbuilding up more localized biotin. Finally, the “amplified” biotindeposit is detected with streptavidin attached to a fluorescentmolecule. Alternatively, the amplified biotin deposit can be detectedwith avidin-peroxidase complex, which is then contacted with DAB toproduce a brown color. In other examples, signal amplification includescontacting the sample with hydrogen peroxide and a tyramide-HQ conjugateafter contacting the sample with an HRP-conjugated tertiary antibodyunder conditions sufficient for depositing HQ at or near the site of theprimary antibody bound to the sample. The sample is then contacted withan enzyme-conjugated antibody (such as an HRP- or AP-conjugatedantibody) that specifically binds to HQ. In some examples, thisenzyme-conjugated antibody is the same as the HRP-conjugated tertiaryantibody. In other examples, the enzyme-conjugated antibody is adifferent antibody than the HRP-conjugated tertiary antibody. The sampleis then contacted with one or more reagents that produce a detectablereaction product in the presence of the enzyme. In some examples, thesample is contacted with an HRP substrate (such as hydrogen peroxide)and a chromogen (such as DAB) that produces a visually detectableproduct in the presence of HRP. In some examples, signal amplificationis carried out using VENTANA OptiView Amplification Kit (Ventana MedicalSystems, Inc., Catalog No. 760-099).

It is also understood that any of the above methods for diagnosis and/ordetection may be carried out using an immunoconjugate of the invention,as described above, in place of or in addition to an unconjugatedanti-OX40 antibody.

F. Biological Samples

In certain embodiments, the anti-OX40 antibodies of the invention (e.g.,SP197) can be used to detect the presence of OX40 in biological samplesusing methods known in the art or described herein.

In some instances a biological sample includes a tissue or a cellsample. For example, a biological sample may include a cell or tissuefrom normal or cancer patients, such as, for example, normal andcancerous tissue of breast, colon, lung, kidney, bone, brain, muscle,stomach, pancreas, bladder, ovary, uterus, as well as heart, embryonic,and placental tissue.

In certain instances the source of the tissue or cell sample may besolid tissue as from a fresh, frozen and/or preserved organ or tissuesample or biopsy or aspirate; blood or any blood constituents; bodilyfluids such as cerebral spinal fluid, amniotic fluid, peritoneal fluid,or interstitial fluid; cells from any time in gestation or developmentof the subject. In some embodiments the biological sample is obtainedfrom in vitro tissue or cell culture. Examples of biological samplesherein include, but are not limited to, tumor biopsies, circulatingtumor cells, serum or plasma, circulating plasma proteins, asciticfluid, primary cell cultures or cell lines derived from tumors orexhibiting tumor-like properties, as well as preserved tumor samples,such as formalin-fixed, paraffin-embedded (FFPE) tumor samples or frozentumor samples.

In some embodiments the biological sample contains compounds which arenot naturally intermixed with the tissue in nature such aspreservatives, anticoagulants, buffers, nutrients, antibiotics, or thelike. In certain embodiments the biological sample has been exposed toand/or contains one or more fixatives. Fixatives that can be used withmethods and compositions of the invention include formalin,glutaraldehyde, osmium tetraoxide, acetic acid, ethanol, acetone, picricacid, chloroform, potassium dichromate and mercuric chloride and/orstabilizing by microwave heating or freezing.

In some embodiments, the biological sample is from a subject having,predisposed to, or being tested for an autoimmune disease. In certainembodiments, the autoimmune disease is an autoimmune rheumatologicdisorder (including rheumatoid arthritis, Sjogren's syndrome,scleroderma, lupus such as SLE and lupus nephritis,polymyositis-dermatomyositis, cryoglobulinemia, anti-phospholipidantibody syndrome, and psoriatic arthritis), an autoimmunegastrointestinal and liver disorder (including inflammatory boweldiseases (e.g., ulcerative colitis and Crohn's disease), autoimmunegastritis and pernicious anemia, autoimmune hepatitis, primary biliarycirrhosis, primary sclerosing cholangitis, and celiac disease),vasculitis (including ANCA-negative vasculitis and ANCA-associatedvasculitis, including Churg-Strauss vasculitis, Wegener'sgranulomatosis, and microscopic polyangiitis), an autoimmuneneurological disorder (including multiple sclerosis, opsoclonusmyoclonus syndrome, myasthenia gravis, neuromyelitis optica, Parkinson'sdisease, Alzheimer's disease, and autoimmune polyneuropathies), a renaldisorder (including glomerulonephritis, Goodpasture's syndrome, andBerger's disease), an autoimmune dermatologic disorder (includingpsoriasis, urticaria, hives, pemphigus vulgaris, bullous pemphigoid, andcutaneous lupus erythematosus), a hematologic disorder (includingthrombocytopenic purpura, thrombotic thrombocytopenic purpura,post-transfusion purpura, and autoimmune hemolytic anemia),atherosclerosis, uveitis, an autoimmune hearing disease (including innerear disease and hearing loss), Behcet's disease, Raynaud's syndrome,organ transplant, or an autoimmune endocrine disorder (includingdiabetic-related autoimmune diseases such as insulin-dependent diabetesmellitus (IDDM), Addison's disease, and autoimmune thyroid disease(including Graves' disease and thyroiditis)).

In other embodiments, the biological sample is from a subject having,predisposed to, or being tested for cancer. In certain embodiments thecancer is carcinoma, lymphoma (including Hodgkin's and non-Hodgkin'slymphoma), blastoma, sarcoma, leukemia, squamous cell cancer, small-celllung cancer, non-small cell lung cancer, adenocarcinoma of the lung,squamous carcinoma of the lung, cancer of the peritoneum, hepatocellularcancer, gastrointestinal cancer, pancreatic cancer, glioma, cervicalcancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breastcancer, colon cancer, colorectal cancer, endometrial or uterinecarcinoma, salivary gland carcinoma, kidney cancer, liver cancer,prostate cancer, vulval cancer, thyroid cancer, hepatic carcinoma,leukemia and other lymphoproliferative disorders, or various types ofhead and neck cancer. In a specific embodiment, the cancer is selectedfrom non-small cell lung cancer (NSCLC), bladder cancer, renal cellcarcinoma (RCC), ovarian cancer, colorectal cancer, triple negativebreast cancer (TNBC), and melanoma.

III. EXAMPLES

The following are examples of methods and compositions of the invention.It is understood that various other embodiments may be practiced, giventhe general description provided above.

Example 1. Generation of Anti-OX40 Antibodies

Anti-OX40 rabbit monoclonal antibodies were generated as schematicallydepicted in FIG. 1. Briefly, the peptide fragment of amino acid residues266-277 of was synthesized. The 12-amino acid fragment intended forimmunization was conjugated to keyhole limpet hemocyanin (KLH), anextensively used carrier protein for stimulating a substantial immuneresponse via antibody production. New Zealand White rabbits wereimmunized with KLH conjugated OX40 antigen emulsified with completeFreund's adjuvant followed by a series of OX40 antigen boosteremulsified with incomplete Freund's adjuvant. The antibody-expressingcells were screened by enzyme-linked immunoabsorbant assay (ELISA) usingthe OX40 antigen. All ELISA positive clones were further screened byimmunohistochemistry (IHC), and the clone producing the antibody withthe highest specificity was selected. For recombinant production ofanti-OX40 antibodies, cDNA coding for the heavy chain and light chainsequences of the antibodies were cloned, expressed by co-transfection,and screened for binding to OX40 by IHC. Anti-OX40 monoclonal antibodySP197 was produced using these methods and subsequently purified byProtein A affinity chromatography. The heavy and light chain variableregion sequences of the SP197 antibody are as follows.

The amino acid sequence of the heavy chain variable region is thefollowing (HVR sequences underlined):

(SEQ ID NO: 16) QSLEESGGRLVAPGGSLTLTCTVSGIDLSSDNIQWVRQAPGKGLEWIGAVDYNNKPFYANWAKGRFTISKTSSTTVDLKMTSL TTEDTATYFCAKNTFSPWGPGTLVTVSS

The amino acid sequence of the light chain variable region is thefollowing (HVR sequences underlined):

(SEQ ID NO: 17) DPAMTQTPSSTSAAVGGTVTINCQSSQSVYNANHLSWFQQKPGQPPKRLIYYISTPDSGVPPRFSGSGSGTQFTLTISGVQCDDA ATYYCAALNSDEVFTFGGGTEVVVK

Example 2. Diagnostic Uses of Anti-OX40 Antibodies

Specific immunohistochemical (IHC) staining of rabbit anti-human OX-40monoclonal antibody (SP197) on control cells and FFPE tissues has beendemonstrated. IHC was performed using StdCC1 cell conditioning withultraView Universal DAB Detection Kit (Ventana Medical Systems, Inc.,Tucson, Ariz.). Primary antibody was incubated for 16 min at 37° C. on aBenchMark ULTRA automated slide stainer (Ventana Medical Systems, Inc.,Tucson, Ariz.). Results are shown at Fig. (A) Mock transfected cells(negative control cells); (B) OX-40 transfected cells (positive controlcells); (C) Reactive lymph node; and (D) Prostate adenocarcinoma. Strongmembrane staining was seen in positive control cells (B) and activatedT-cells in reactive lymph node and prostate adenocarcinoma.

OTHER EMBODIMENTS

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, the descriptions and examples should not be construed aslimiting the scope of the invention. The disclosures of all patent andscientific literature cited herein are expressly incorporated in theirentirety by reference.

The invention claimed is:
 1. An isolated antibody capable ofspecifically binding to a C-terminal portion of OX40 comprising aminoacids 266-277 of SEQ ID NO: 1, wherein the antibody comprises a heavychain variable domain (VH) sequence comprising the following heavy chainhypervariable regions (HVR-H): (a) an HVR-H1 comprising the amino acidsequence of SEQ ID NO: 2; (b) an HVR-H2 comprising the amino acidsequence of SEQ ID NO: 3; (c) an HVR-H3 comprising the amino acidsequence of SEQ ID NO: 4; and and a light chain variable domain (VL)sequence comprising the following light chain hypervariable regions(HVR-L): (d) an HVR-L1 comprising the amino acid sequence of SEQ ID NO:9; (e) an HVR-L2 comprising the amino acid sequence of SEQ ID NO: 10;and (f) an HVR-L3 comprising the amino acid sequence of SEQ ID NO: 11.2. The antibody of claim 1, wherein the VH sequence further comprisesthe following heavy chain variable domain framework regions (FR-Hs): (g)FR-H1 comprising the amino acid sequence of SEQ ID NO: 5; (h) FR-H2comprising the amino acid sequence of SEQ ID NO: 6; (i) FR-H3 comprisingthe amino acid sequence of SEQ ID NO: 7; and (j) FR-H4 comprising theamino acid sequence of SEQ ID NO:
 8. 3. The antibody of claim 1, whereinthe VL sequence further comprises the following light chain variabledomain FRs: (k) FR-L1 comprising the amino acid sequence of SEQ ID NO:12; (l) FR-L2 comprising the amino acid sequence of SEQ ID NO: 13; (m)FR-L3 comprising the amino acid sequence of SEQ ID NO: 14; and (n) FR-L4comprising the amino acid sequence of SEQ ID NO:
 15. 4. The antibody ofclaim 1, wherein: the VH sequence has at least 95% sequence identity tothe amino acid sequence of SEQ ID NO: 16; or the VL sequence has atleast 95% sequence identity to the amino acid sequence of SEQ ID NO: 17;or the VH sequence has at least 95% sequence identity to the amino acidsequence of SEQ ID NO: 16 and the VL sequence has at least 95% sequenceidentity to the amino acid sequence of SEQ ID NO:
 17. 5. The antibody ofclaim 1, wherein the VH sequence comprises SEQ ID NO:
 16. 6. Theantibody of claim 1, wherein the VL sequence comprises SEQ ID NO:
 17. 7.The antibody of claim 1, wherein the antibody further comprises thefollowing heavy chain variable domain framework regions (FR-Hs) andlight chain variable domain framework regions (FR-Ls): (g) FR-H1comprising the amino acid sequence of SEQ ID NO: 5; (h) FR-H2 comprisingthe amino acid sequence of SEQ ID NO: 6; (i) FR-H3 comprising the aminoacid sequence of SEQ ID NO: 7; (j) FR-H4 comprising the amino acidsequence of SEQ ID NO: 8; (k) FR-L1 comprising the amino acid sequenceof SEQ ID NO: 12; (l) FR-L2 comprising the amino acid sequence of SEQ IDNO: 13; (m) FR-L3 comprising the amino acid sequence of SEQ ID NO: 14;and (n) FR-L4 comprising the amino acid sequence of SEQ ID NO:
 15. 8.The antibody of claim 1, wherein the antibody comprises a VH sequence ofSEQ ID NO: 16 and a VL sequence of SEQ ID NO:
 17. 9. The antibody claim1, wherein the antibody is a monoclonal antibody.
 10. The antibody ofclaim 9, wherein the monoclonal antibody is a rabbit monoclonalantibody.
 11. The antibody of claim 1, wherein the antibody is an IgGantibody.
 12. The antibody of claim 1, wherein the antibody is anantibody fragment that specifically binds OX40.
 13. The antibody ofclaim 12, wherein the antibody fragment is selected from the groupconsisting of Fab, single chain variable fragment (scFv), Fv, Fab′,Fab′-SH, F(ab′)2, and diabody.
 14. A kit comprising an antibodyaccording to claim
 1. 15. The kit of claim 14, further comprising asecondary antibody immunoreactive with the antibody capable ofspecifically binding to the C-terminal portion of OX40.
 16. The kit ofclaim 15, further comprising an enzyme coupled to or adapted to becoupled to the secondary antibody.
 17. The kit of claim 16, furthercomprising a set of reagents reactive with the enzyme to deposit achromogen or fluorophore.
 18. The kit of claim 17, wherein the set ofreagents reactive with the enzyme comprises a tyramide conjugate.
 19. Amethod of detecting the presence or expression level of OX40 in abiological sample comprising contacting the biological sample with theantibody of claim 1 and detecting the presence of the bound antibody.20. The method of claim 19, wherein the detecting is byimmunohistochemistry (IHC), immunofluorescence, or immunoblot.
 21. Themethod of claim 20, wherein the detecting is by IHC.
 22. The method ofclaim 19, wherein the sample comprises a fixed tissue.
 23. The method ofclaim 22, wherein the fixed tissue is a FFPE tissue.
 24. The method ofclaim 19, wherein the sample is from a subject having or predisposed tocancer or autoimmune disease.